Abstract The objective of this project is to develop a multi-dose safety profile for a novel drug candidate to treat a devastating neurodegenerative disease called Spinal Muscular Atrophy (SMA). SMA is an autosomal recessive disorder that is the leading genetic cause of infantile death, occurring in ~1:6,000 live births. The gene responsible for SMA is called survival motor neuron-1 (SMN1). SMN2 is nearly identical to SMN1, however, mutations in SMN2 have no clinical consequence if SMN1 is retained. The reason SMN2 cannot prevent disease development in the absence of SMN1 is that most SMN2-derived transcripts are alternatively spliced, resulting in a truncated and unstable protein. The presence of SMN2 offers exciting therapeutic strategies including modulating the pathogenic alternative splicing of SMN2 exon 7. In this project, we will build upon the findings in the Lorson lab at the University of Missouri that identified ?Element 1? (E1) as a potent repressor of SMN2 exon 7 inclusion. They discovered a single Phosphorodiamidate Morpholino Oligomer (PMO), an antisense oligo (ASO) targeting Element 1 significantly extended survival in two important SMA animal models. Rigorous analysis of Element 1 sequence led to the design of several additional PMOs, some of which simultaneously targeted both ends of the 51-nucleotide E1. A panel screen of these ASOs identified a lead candidate exhibiting greater efficacy across a range of doses examined in cellular and SMA animal models. This compound, a single 20-base PMO called E1v1.11, is the focus of this Phase I proposal. The Lorson lab has collected data from single-dose, dose-range studies with E1v1.11 in SMA mice showing single doses that extend survival of SMA mice more than 140 days. However, repeat dosing is necessary for treatment in humans. Thus, Shift proposes a pilot safety study modeled from both FDA guidelines and pre-clinical studies for Spinraza (first FDA approved SMA drug), to prepare for Investigational New Drug (IND)-enabling studies. As part of the investigational drug process, FDA requires safety data from two modes of delivery, and adult and juvenile animals for drugs to treat pediatric patients. In Aim 1, we will collect toxicology data in a multi-dose study of intrathecal injections of E1v1.11 PMO in adult Sprague-Dawley rats. Aim 2 will collect toxicology data in a multi-dose study of subcutaneous injections of E1v1.11 PMO in juvenile Sprague- Dawley rats. This project brings the exceptional properties of PMOs (efficacy, solubility, tolerability) to an entirely unique genetic target within SMN2, the E1 repressor region. SMA is a complex genetic disorder with a broad clinical spectrum. With the recent FDA approval of Spinraza, it is important to continue to develop additional unique SMA therapeutics. We believe an E1v1.11 PMO will be a powerful and valuable addition to the SMA drug portfolio.